Acetylcholine (ACh) is a neurotransmitter widely used throughout the brain and body to subserve functions critical to human health, including cardiovascular and autonomic health. Dysfunctional brain ACh signaling may also contribute to the cognitive deficits that accompany many psychiatric conditions, including ADHD, depression, schizophrenia, and dementia, yet the cellular processes that regulate cholinergic tone remain poorly understood. ACh levels are restricted by the limited supply of the precursor molecule choline, which must be actively transported into nerve terminals by the high-affinity choline transporter (CHT). Our lab has recently characterized novel transgenic mouse models of both decreased (CHT+/-) and increased (BAC-CHT) CHT protein expression (Ferguson et al., 2004; Bazalakova et al., 2007; English et al., 2010; Lund et al., 2010; Holmstrand et al., 2011). Alterations to choline uptake, tissue ACh content and ACh release in these animals suggest that CHT may also impact the distribution of synaptic vesicles to physiologically distinct pools. Using optical monitoring of synaptic vesicle release and endocytosis, the experiments outlined in this application will determine if the relative protein levels of CHT are associated with proportional changes to the size of the reserve pool of cholinergic synaptic vesicles. Additional experiments will use viral vectors to induce overexpression of wt (wild type) and mutant CHT isoforms to determine the effects of acute overexpression of human CHT and investigate the role of an intracellular endocytic motif present in the CHT protein sequence in determining synaptic vesicle pool identity. This work will provide me with extensive training in advanced cellular neurobiology techniques and possibly establish a novel role for CHT in the control of regulated neurotransmitter release. PUBLIC HEALTH RELEVANCE: Acetylcholine (ACh) is used at synapses throughout the nervous system and impacts a wide range of physiological processes, including voluntary muscle contraction, cardiovascular function, digestion, and cognition. The high-affinity choline transporter (CHT) influences the amount of ACh available for release by providing the rate-limiting precursor choline for ACh synthesis, but may also play a role in regulating vesicle release properties at the synapse. The proposed project will: 1) determine if CHT can alter the distribution of synaptic vesicles between the readily releasable pool of synaptic vesicles and the release-resistant reserve pool; and 2) examine the role of an endocytic motif in the CHT protein sequence in directing synaptic vesicle sorting to these distinct physiological pools.